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Studies in Glycogen Storage Disease. IV. Leukocyte phosphorylase in a family with type VI GSD
Studies in Glycogen Storage Disease. IV. Leukocyte Phosphorylase in a Family with Type VI GSD By DAVID SCHWARTZ, MICHAEL SAWN, ALLAN DRASH AND JAMES FIELD 4 patient with Type VI Glycogen Storage Disease and members of his family have been studied utilizing leukocyte phosphorylase activities and erythrocyte glycogen levels to help elucidate tbe mode of inheritance of this disease. Leukocyte phospborylase activities were found to be low in the patient, his clinically normal brother, parents, maternal grandparents, paternal grandmother, two maternal uncles and a paternal aunt, suggesting an autosomal recessive mode of inheritance in this family. Other investlgators have described both dominant and recessive modes of inheritance in Type VI GSD. Recent studies have indicated the existence of two forms of this disease,
phosphorylase deficiency and phosphorylase kinase deficiency. Although phosphorylase kinase activities were not measured in this family, there is suggestive evidence that our patient has phosphorylase deficiency with a normal activating system. It is suggested that Type VI GSD associated with phosphorylase kinase deficiency follows a dominant pattern of inheritance, while the form of this disease associated with phosphorylase deficiency follows a recessive pattern of inheritance. The erythrocyte glycogen levels proved to be an unsatisfactory index of the heteroxygous state. (Metabolism 19: No. 3, March, 23&245, 1970)
HE MODE OF INHERITANCE of hepatic phosphorylase deficiency (Type VI glycogen storage disease) has not been clearly elucidated. Type VI glycogen storage disease (GSD) was first described by Hers1 in 1959 when he reported three children whose hepatic phosphorylase activities were between 20 per cent and 25 per cent of normal. In 1961 two groups of investigators, Williams and Field” and Hiilsman, Oei and van Creveld” reported low leukocyte phosphorylase in five patients with proven hepatic phosphorylase deficiency. Low values of leukocyte phosphorylase activity were also found in asymptomatic relatives of these patients suggesting the possibility of identifying heterozygotes for Type VI GSD. A dominant mode of inheritance for this disease was postulated on the basis of decreased enzyme activity found in only one of the parents in each family. In 1966 Wallis and his co-workers4 studied another
T
From the Clinical Research Unit and the Departments of Medicine and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication September 1, 1969. This investigation was supported by USPHS Grant AM-08333 and General Clinical Research Center Grants FR-56 and FR-84 from the National Institutes of Health. Presbyterian-University Hospital, Pittsburgh, Pa.; DAVID SCHWARTZ, M.D.: Resident, formerly fourth-year medical student, University of Pittsburgh School of Medicine, MICHAEL SAVIN, M.D.: Resident, Duke University Hospital, Durham, N. C.; formerly fourth-year medical student, University of Pittsburgh School of Medicine. ALLAN DRASH, M.D.: Associate Professor of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pa.; JAMES FIELD, M.D. : Director, Clinical Research Unit, Presbyterian-University Hospital; Professor of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 238
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family with Type VI GSD. In this family both parents (clinically asymptomatic) had low leukocyte phosphorylase activities and high erythrocyte glycogen levels, suggesting an autosomal recessive mode of inheritance. These workers also measured erythrocyte glycogen levels in the family previously reported by Williams and Field.2 Erythrocyte glycogen levels were elevated in the mother, but normal in the father. These results were in agreement with the suggestion of Williams and Field that this family demonstrated a dominant pattern of inheritance. These two different types of inheritance suggested two distinct forms of Type VI GSD.4 Evidence for a second variety of Type VI GSD was provided by Hug’s observation of patients with low hepatic phosphorylase secondary to abnormalities in the phosphorylase activating system rather than to a deficiency of phosphorylase per se.” Further evidence was provided by Huijinga who measured leukocyte phosphorylase kinase activities in eleven patients with low leukocyte phosphorylase activities and found a deficiency of the kinase in ten of these patients. Studies of family members of these patients suggested a sexlinked mode of inheritance for the phosphorylase kinase deficiency and an autosomal recessive inheritance for the phosphorylase deficiency. In the present study leukocyte phosphorylase activities and erythrocyte glycogen content have been examined in another patient with Type VI GSD and in members of his family to learn more about the inheritance of this disease. CASE REPORT The patient, T.R., a white male, was the product of a 37-week gestation, during which time his mother gained 43 pounds and had ankle edema, but no hypertension or proteinuria. His birth weight was 5 lb. 11 oz.; his delivery and neonatal period were uncomplicated. He was breast fed until four months of age and was begun on cereal at two weeks of age. At four months of age he was placed on whole milk. From age four to six months the patient’s mother noted a gradual increase in the size of the abdomen and at age six months the family physician noted an enlarged liver. He was admitted to another hospital where he was said to have had a “high blood sugar”, and he was transferred to Children’s Hospital of Pittsburgh for further evaluation. Upon his admission to Children’s Hospital his physical examination revealed him to be well developed and well nourished with a distended, tympanitic abdomen. The liver was palpable 9 cm below the right costal margin and was described as soft and smooth with a rounded edge. The spleen was not palpable. He had bilateral inguinal hernias and a capillary hemangioma on the anterior aspect of the right thigh. The remainder of his physica examination was within normal limits. His hemoglobin was 10 Gm. per cent, hematocrit 35 per cent and white blood count 15,000 with a normal differential. Routine urinalysis was normal; no acetonuria was present. Fasting blood sugars were 63 mg. per cent and 70 mg. per cent on two occasions. Uric acid was 4.1 mg. per cent and cholesterol was 133 mg. per cent. BUN, electrolytes, alkaline phosphatase, total protein and serum protein electrophoresis were all within normal limits. X-ray studies of the chest and skull were normal; a skeletal survey revealed no abnormalities. A glucagon tolerance test, using 0.03 mg. glucagon/Kg. body weight subcutaneously, showed a fasting blood sugar of 63 mg. per cent, at 20 min. 97 mg. per cent, 40 min. 78 mg. per cent, 60 min. 90 mg. per cent, 80 min. 77 mg. per cent, 100 min. 66 mg. per cent, and at 120 min. 69 mg. per cent. Liver biopsy was performed on the eighth hospital day. The parenchymal cells were increased and quite variable in size and sinusoids were not evident. There were many large vacuoles present which were shown to contain glycogen by the PAS and PAS diastase reaction. Quantitative glycogen determination and enzyme assays were not done. He was discharged with a diagnosis of “Glycogen Storage Disease.”
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Over the ensuing years he was treated with frequent feedings. He had weekly episodes of vomiting occurring in the morning if he drank milk, and at other times when he became excited or angry. He also had frequent episodes of otitis and tonsillitis. The mother reported no convulsions or muscle weakness in the child. In August 1968 at age 6% years the patient was re-admitted to Children’s Hospital for further investigation. At this time it was noted that his developmental milestones were normal and that he was a “superior” student in school. His physical examination revealed normal vital signs. His height was 112 cm. (tenth percentile), weight 21.2 Kg. (25th percentile), head circumference 54 cm. (97th percentile), chest circumference 61 cm. (90th percentile), and abdominal girth 67.5 cm. (> 97th percentile). A grade II/VI apical systolic murmur was heard and it radiated widely. His liver was enlarged to 10 cm. below the right costal margin; it was nontender, smooth and had a rounded edge. The remainder of his physical examination was unremarkable. Admission laboratory studies, including hemoglobin and hematocrit, white blood count and differential, bleeding time, clotting time, BUN, electrolytes, SGOT, SGPT, serum protein electrophoresis and blood gases were all normal. A single urinalysis at the time of admission revealed 3 plus acetonuria, but was otherwise normal. Skeletal survey revealed a bone age of four years, which was retarded for his chronological age. Blood sugars obtained four hours after meals were 92 mg. per cent, 80 mg. per cent, 79 mg. per cent and 87 mg. per cent. An oral GTT (1.8 Gm. glucose/Kg.) revealed a fasting blood sugar of 66 mg. per cent, at 30 min. 148 mg. per cent, 60 min. 162 mg. per cent, 120 min. 124 mg. per cent, 180 min. 59 mg. per cent, 240 min. 58 mg. per cent and at 300 min. 67 mg. per cent. A glucagon tolerance test (0.03 mg. glucagon/Kg.. subcutaneously) gave the following results: fasting blood glucose 67 mg. per cent, 10 min. 80 mg. per cent, 20 min. 96 mg. per cent, 30 min. 113 mg. per cent, 45 min. 117 mg. per cent, 60 min. 104 mg. per cent. 90 min. 79 mg. per cent and 120 min. 70 mg. per cent. Liver and muscle biopsies were performed on the patient, and the results of biochemical analysis are tabulated in Table I.
METHODS AND MATERIALS The methods for assay of leukocyte phosphorylase,” hepatic and muscle phosphorylase,r hepatic glucose-6-phosphatase ,r hepatic and muscle debranching enzyme,7 and hepatic and muscle alpha-glucosidases have been previously described. The methods for assay of glycogen in liver,? muscle,9 and erythrocytesla are also described elsewhere. Previously Williams and Field reported normal leukocyte phosphorylase to be 29.6 + 1.2 (SEM) pg. PO,/107 wbc/30 min. However, using the exact procedure we have found leukocyte phosphorylase activity to be 73.6 t 3.0 (SEM) &g. PO,/107 wbc/30 min. in a series of 26 normals. The range was from 52.0 to 106.8. The reason for the difference between these two normal series is not apparent. Esmann et al, 11 however, utilizing this same assay, recently reported activities of leukocyte phosphorylase in normals with values similar to ours (73.28 ? 1.09 (SEM) pg. PO,/107 wbc/30 min.). The reliability of the erythrocyte glycogen assay was tested by determining the recovery of 100 fig. of glycogen added to aliquots of packed erythrocytes. The recoveries ranged from 19 per cent to 35 per cent. The results were not corrected for recovery. RESULTS
The diagnosis of Type VI GSD in the patient was established by the clinical findings and by the marked reduction in liver phosphorylase (20 min. 12.8 mg. POJGm. protein and 30 min. 18.0 mg. POJGm. protein) and in leukocyte phosphorylase 35.98 PGm. P04/107wbc/30 min.). Liver glycogen was increased (7.5% ), whereas muscle glycogen content was normal (0.5% ). Other liver and muscle enzymes concerned with glycogen metabolism were normal. Leukocyte phosphorylase and erythrocyte glycogen values in the patient and eleven of his relatives are summarized in Table 2 and the pedigree illustrated in Fig. 1. The patient was the only member of his family with signs and symptoms
Debranching (14C method) Glycogen alpha-Glucosidase Phosphorylase
Debranching (14C method) Phosphorylase Glycogen
Liver Muscle Muscle Muscle
Muscle WBC RBC
wbc: white blood cells. rbc: red blood cells. cpm: counts per minute.
Glycogen alpha-Glucosidase Glucose-6-phosphatase Phosphorylase
Assay
Liver Liver Liver Liver
Tissue
7.5% 6.89 24 12.8 (20 min.) and 18.0 (30 min.) 1,340,000 0.5% 0.90 263 (20 min.) and 362 (30 min.) 1,000,000 35.98 60.25
Results
Table 1 .-Enzyme
Assays (Mean C SEM)
up to 5% 12.93 t 2.37 w moles glucose liberated/min./mg. protein 26.7 * 5.1 mg. phosphorus liberated/Gm. protein 72.0 f 14.7 (20 min.) and 81.0 t 18.9 (30 min.) mg. phosphorus liberated/ Gm. protein 1,123,OOO k 435,000 cpm/Gm. protein up to 1.0% 1.74 k 41 mp moles glucose liberated/min./mg. protein 144 f 21 (20 min.) and 220 + 37 (30 min.) mg. phosphorus liberated/Gm. protein 1,307,OOO + 103,000 cpm/Gm. protein 73.61 -t 3.01 pg. PO,/107 wbc/30 min. 23.59 k 5.94 ,eg. glycogen/Gm. Hgb.
Normals
and Special Studies on Patient T.R.
N P
? $
u i;3
F $
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Table 2.-Leukocyte Phosphorylase Activities and Eerythrocyte Glycogen Levels in Normal Subjects, Patient T.R., and Members of His Family ___~~_. Erythrocyte Glycogen .__@g. GlycogedGm. Hgb.
Leukocyte Phosphorylase Person
Patient (T.R.) Brother (J.R.) Mother (CR.) Father (C.L.R.) Grandmother (M.R.C.) (maternal) Grandfather (R.C.) (maternal) Grandmother (H.R.) (paternal) Uncle, (L.C.) (maternal) Uncles (J.C.) (maternal) Aunt (G.S.) (paternal) Grandaunt (E.G.) (maternal) Granduncle (J.A.C.) (maternal) Mean Standard Error of the Mean Normals Range Mean Standard Error of the Mean *Number
____~~~ k%. PO+/lO’wbc/30 min.
35.98 33.68 25.26 45.12 50.68 47.20 44.25 22.31 24.51 44.36 72.20 74.96 43.38 4.91 (n = 26) 52.01-106.81 73.61 3.02
60.25
(2)* (1) (2) (1) (4) (2) (1) (2) (2) (1) (1) (2)
(l)*
117.56 (2) 39.49 (1) 33.30 (3) 13.66 (2) 42.42 (1) 20.33 (2) 66.51 (2) 71.65 (1) 22.49 (1) 48.77 9.89 (n = 21) 3.91-133.16 23.59 5.94
of determinations.
of glycogen storage disease. The patient’s leukocyte phosphorylase activity determined on two occasions averaged 35.98 pg. POa/107wbc/30 min., well outside our normal range. Both the mother’s leukocyte phosphorylase activity (25.26 pg. P01/107wbc/30 min.) and the father’s leukocyte phosphorylase activity (45.12 pg. P0,/107wbc/30 min.) were reduced. The patient’s brother had a leukocyte phosphorylase activity similar to that of the patient (33.68 pg. POJlO’wbc/30 min.). He was asymptomatic and did not have hepatomegaly. The patient’s maternal grandmother had a leukocyte phosphorylase activity just below our normal range 50.68 pg. P01/107wbc/30 min.). The maternal grandfather’s leukocyte phosphorylase activity (47.2 pg. PO,/lOrwbc/ 30 min.) was also outside our normal range. The only paternal grandparent studied was the paternal grandmother. Her leukocyte phosphorylase activity was 44.25 pg. P0+/107wbc/30 min., which also falls below our normal range. Two maternal uncles and one paternal aunt had low leukocyte phosphorylase activities. The maternal grandfather’s brother and sister had normal activities of this enzyme. The patient’s leukocyte phosphorylase activity did not differ significantly from the activities found in other family members whose values fell below our normal range. Table 2 also includes the results of the erythrocyte glycogen studies. The 2 1 normal subjects had values ranging from 3.91 pg. glycogen/Gm. Hgb to 133.16 pg. glycogen/Gm. Hgb, with a mean of 23.59 pg. glycogen/Gm.Hgb. The patient’s erythrocyte glycogen (60.3 pg./Gm. Hgb) and the erythrocyte glycogen levels of all members of his family tested were within our normal range. DISCUSSION
This patient provides another
example of reduced
hepatic
and leukocyte
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EG.
7220
-
7%
Cs.
44.36
Demo& LeukocyteFt-qhcfyhse DecreasedLeuk and Clinkd hkZ%Eb~yk Type VI GSD
q=Male o=Femde NT= Not Tested Fig. 1 .-Pedigree
of family studied.
phosphorylase activity found in patients with Type VI GSD. Similar observations of reduced leukocyte phosphorylase activity in patients with this disease have been made by other workers.2-4*6J1J2 In the present family reduced leukocyte phosphorylase activity was found in the patient’s mother and father, brother, the paternal grandmother, maternal grandparents, two maternal uncles and a paternal aunt, none of whom have clinical evidence of glycogen storage disease. A sex-linked mode of inheritance for this disease is unlikely because the patient’s brother, father, maternal grandfather, and maternal uncle have low leukocyte phosphorylase activities and no clinical evidence of glycogen storage disease. A dominant mode of inheritance for this disease is also unlikely since none of the relatives of the patient who have low leukocyte phosphorylase activities have any clinical evidence of Type VI GSD. Therefore, an autosomal recessive mode of inheritance is most likely. This hypothesis is supported by the finding of low leukocyte phosphorylase activities in clinically normal relatives of both sexes on either side of the family, this finding being the only abnormality manifested by the heterozygous individual. Both an autosomal recessive and a sex-linked inheritance have been postulated for Type VI alycogen storage disease. Williams and Field* in 1961 re-
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ported decreased activities of leukocyte phosphorylase in two brothers with Type VI GSD. Leukocyte phosphorylase activity in the patient’s mother was intermediate between normal values and those of her sons while the father was normal. Similar findings were reported by Htilsman et al.” in a family they studied. The findings of these two groups of investigators suggest a dominant mode of inheritance, either sex-linked or autosomal. Hers,l however, in his original description of this disease, reported two females with clinical and laboratory findings of Type VI GSD whose parents had no clinical evidence of glycogen storage disease, thus mitigating against a sex-linked mode of inheritance. Wallis et a1.4 found markedly decreased leukocyte phosphorylase activity in a patient with Type VI GSD and levels intermediate between those of the patient and normal subjects in both parents, the maternal grandmother and the maternal grandmother’s mother, all of whom were clinically normal. On the basis on these results it was concluded that this family demonstrated an autosomal recessive mode of inheritance. Esmann et al.ll found low leukocyte phosphorylase levels in a patient with Type VI GSD, his brother, mother. maternal grandfather and a distant male cousin on the maternal side, whereas the father had normal leukocyte phosphorylase activity. The authors suggested a sex-linked mode of inheritance. HuijingO studied leukocyte phosphorylase and leukocyte phosphorylase kinase activities in 11 patients with Type VI GSD. Ten of these patients (9 males and 1 female) had low activities of leukocyte phosphorylase kinase (0 to 60% of the lowest normal values). Five mothers of the children with phosphorylase kinase deficiency were tested; four had low leukocyte phosphorylase kinase activities. Three fathers were also tested and found to have normal leukocyte phosphorylase kinase activities. Two of these three fathers were from families in which the mother’s leukocyte phosphorylase kinase was found to be low. He suggested that these results were best explained by postulating a sex-linked mode of inheritance in the form of Type VI GSD associated with a deficiency of leukocyte phosphorylase kinase (Type VI A) .13 The eleventh patient had low leukocyte phosphorylase activity, but normal leukocyte phosphorylase kinase activity. The mother and a sib of the patient also had normal leukocyte phosphorylase kinase activity. Both the mother and father of this patient, however, had low leukocyte phosphorylase activities suggesting an autosomal recessive mode of inheritance in the form of Type VI GSD associated with a deficiency of leukocyte phosphorylase in the presence of an intact activating system (Type VI B) .I3 Since our patient’s family appears to demonstrate an autosomal recessive pattern, he probably has a deficit of phosphorylase with an intact activating system. Huijing” noted that the addition of 1 mM AMP restored to normal leukocyte phosphorylase activity of patients with leukocyte phosphorylase kinase deficiency. Although leukocytes from our patient were assayed in a medium containing 0.8 mM AMP (close to that used by Huijing). phosphorylase activity was still significantly decreased. This suggests that our patient has a deficiency of phosphorylase itself with an intact activating system. It was not possible to assay leukocytes of our patient and his relatives for phosphorylase kinase. In addition, assay of leukocyte phosphorylase with and without added AMP would be of value.
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Although Wallis et al.* felt that elevations of erythrocyte glycogen values were a sensitive index of the carrier state of Type VI GSD, our results were not consistent with this conclusion. They found elevated erythrocyte glycogen levels in their patient, both parents, the maternal grandmother, the maternal grandmother’s mother and in six other members of both sides of the family. However, in our patient and his relatives erythrocyte glycogens were normal. The poor recoveries of glycogen obtained using this determination could certainly be a major factor in the apparent uselessness of this procedure in our hands. SUMMARY
A child with Type VI GSD, confirmed by biopsy, and 12 members of his immediate family have been studied utilizing leukocyte phosphorylase activities and erythrocyte glycogen levels to further elucidate the pattern of inheritance of this disease. Leukocyte phosphorylase activities were low in the patient and his clinically normal brother, parents, maternal grandparents, paternal grandmother, two maternal uncles and a paternal aunt. The data are consistent with an autosomal recessive mode of inheritance in this family. Erythrocyte glycogen studies were not useful in defining the mode of inheritance of this disease. The data are discussed in terms of the two modes of inheritance and the two enzymatic defects which have been described in this disease. ACKNOWLEDGMENTS We would like to thank Dr. Mark Steele for his helpful comments of this paper.
during the preparation
REFERENCES 1. Hers, H. G.: Etudes enzymatiques sur fragments hepatiques. Rev. Intern. Hepatol. 12:35, 1959. 2. Williams, H. E., and Field, J. B.: Low leukocyte phosphorylase in hepatic phosphorylase deficient glycogen storage disease. J. Clin. Invest. 40:1841, 1961. 3. Hiilsmann, W. C., Oei, T. L., and van Creveld, S.: Phosphorylase activity in leukocytes from patients with glycogen storage disease. Lancet 2:581, 1961. 4. Wallis, P. G., Sidbury, J. B., and Harris, R. C.: Hepatic phosphorylase defect: studies on peripheral blood. Amer. I. Dis. Child. 111:278, 1966. 5. Hug, G., Garancis, I. C., Schubert, W. K., and Kaplan, S.: Glycogen storage disease, Types II, III, VII, and IX. Amer. J. Dis. Child., 111:457, 1966. 6. Huijing, F.: Enzymes of glycogen metabolism in leukocytes in relation to glycogen-storage disease. Control of GlyUniversitetsforlaget, cogen Metabolism, 1968, p. 115. 7. Field, J. B., Epstein, S. M., and Egan, T.: Studies in glycogen storage disease. I.
Intestinal glucose-6-phosphatase activity in patients with Von Gierke’s disease and their parents. I. Clin. Invest. 44:1240 ,1965. 8. Williams, H. E.: Alpha-glucosidase in human leukocytes. Biochem. Biophys. Acta. 124~34, 1966. 9. Caroll, N. V., Longley, R. W., and Roe, J. H.: The determination of glycogen in liver and muscle by use of the anthrone reagent. J. Biol. Chem. 220:583, 1956. 10. Williams, H. E., Kendig, E. M., and Field, J. B.: Leukocyte debranching enzyme in glycogen storage disease. J. Clin. Invest. 42:656, 1963. 11. Esmann, V., Hobolth, N., and Jorgensen, J. I.: Heredity of leukocyte phosphorylase and amylo-l , 6-glucosidase deficiency. J. Pediat. 74:90, 1969. 12. van Creveld, S., and Huijing, F.: Glycogen storage disease. Biochemical and clinical data in sixteen cases. Amer. J. Med. 38:554, 1965. 13. van Creveld, S., and Huijing, F.: Glycogenosis. XII Congreso International de Pediatria II, 1968, pp. 554-572.
phosphorylase deficiency and phosphorylase kinase deficiency. Although phosphorylase kinase activities were not measured in this family, there is suggestive evidence that our patient has phosphorylase deficiency with a normal activating system. It is suggested that Type VI GSD associated with phosphorylase kinase deficiency follows a dominant pattern of inheritance, while the form of this disease associated with phosphorylase deficiency follows a recessive pattern of inheritance. The erythrocyte glycogen levels proved to be an unsatisfactory index of the heteroxygous state. (Metabolism 19: No. 3, March, 23&245, 1970)
HE MODE OF INHERITANCE of hepatic phosphorylase deficiency (Type VI glycogen storage disease) has not been clearly elucidated. Type VI glycogen storage disease (GSD) was first described by Hers1 in 1959 when he reported three children whose hepatic phosphorylase activities were between 20 per cent and 25 per cent of normal. In 1961 two groups of investigators, Williams and Field” and Hiilsman, Oei and van Creveld” reported low leukocyte phosphorylase in five patients with proven hepatic phosphorylase deficiency. Low values of leukocyte phosphorylase activity were also found in asymptomatic relatives of these patients suggesting the possibility of identifying heterozygotes for Type VI GSD. A dominant mode of inheritance for this disease was postulated on the basis of decreased enzyme activity found in only one of the parents in each family. In 1966 Wallis and his co-workers4 studied another
T
From the Clinical Research Unit and the Departments of Medicine and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication September 1, 1969. This investigation was supported by USPHS Grant AM-08333 and General Clinical Research Center Grants FR-56 and FR-84 from the National Institutes of Health. Presbyterian-University Hospital, Pittsburgh, Pa.; DAVID SCHWARTZ, M.D.: Resident, formerly fourth-year medical student, University of Pittsburgh School of Medicine, MICHAEL SAVIN, M.D.: Resident, Duke University Hospital, Durham, N. C.; formerly fourth-year medical student, University of Pittsburgh School of Medicine. ALLAN DRASH, M.D.: Associate Professor of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pa.; JAMES FIELD, M.D. : Director, Clinical Research Unit, Presbyterian-University Hospital; Professor of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 238
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family with Type VI GSD. In this family both parents (clinically asymptomatic) had low leukocyte phosphorylase activities and high erythrocyte glycogen levels, suggesting an autosomal recessive mode of inheritance. These workers also measured erythrocyte glycogen levels in the family previously reported by Williams and Field.2 Erythrocyte glycogen levels were elevated in the mother, but normal in the father. These results were in agreement with the suggestion of Williams and Field that this family demonstrated a dominant pattern of inheritance. These two different types of inheritance suggested two distinct forms of Type VI GSD.4 Evidence for a second variety of Type VI GSD was provided by Hug’s observation of patients with low hepatic phosphorylase secondary to abnormalities in the phosphorylase activating system rather than to a deficiency of phosphorylase per se.” Further evidence was provided by Huijinga who measured leukocyte phosphorylase kinase activities in eleven patients with low leukocyte phosphorylase activities and found a deficiency of the kinase in ten of these patients. Studies of family members of these patients suggested a sexlinked mode of inheritance for the phosphorylase kinase deficiency and an autosomal recessive inheritance for the phosphorylase deficiency. In the present study leukocyte phosphorylase activities and erythrocyte glycogen content have been examined in another patient with Type VI GSD and in members of his family to learn more about the inheritance of this disease. CASE REPORT The patient, T.R., a white male, was the product of a 37-week gestation, during which time his mother gained 43 pounds and had ankle edema, but no hypertension or proteinuria. His birth weight was 5 lb. 11 oz.; his delivery and neonatal period were uncomplicated. He was breast fed until four months of age and was begun on cereal at two weeks of age. At four months of age he was placed on whole milk. From age four to six months the patient’s mother noted a gradual increase in the size of the abdomen and at age six months the family physician noted an enlarged liver. He was admitted to another hospital where he was said to have had a “high blood sugar”, and he was transferred to Children’s Hospital of Pittsburgh for further evaluation. Upon his admission to Children’s Hospital his physical examination revealed him to be well developed and well nourished with a distended, tympanitic abdomen. The liver was palpable 9 cm below the right costal margin and was described as soft and smooth with a rounded edge. The spleen was not palpable. He had bilateral inguinal hernias and a capillary hemangioma on the anterior aspect of the right thigh. The remainder of his physica examination was within normal limits. His hemoglobin was 10 Gm. per cent, hematocrit 35 per cent and white blood count 15,000 with a normal differential. Routine urinalysis was normal; no acetonuria was present. Fasting blood sugars were 63 mg. per cent and 70 mg. per cent on two occasions. Uric acid was 4.1 mg. per cent and cholesterol was 133 mg. per cent. BUN, electrolytes, alkaline phosphatase, total protein and serum protein electrophoresis were all within normal limits. X-ray studies of the chest and skull were normal; a skeletal survey revealed no abnormalities. A glucagon tolerance test, using 0.03 mg. glucagon/Kg. body weight subcutaneously, showed a fasting blood sugar of 63 mg. per cent, at 20 min. 97 mg. per cent, 40 min. 78 mg. per cent, 60 min. 90 mg. per cent, 80 min. 77 mg. per cent, 100 min. 66 mg. per cent, and at 120 min. 69 mg. per cent. Liver biopsy was performed on the eighth hospital day. The parenchymal cells were increased and quite variable in size and sinusoids were not evident. There were many large vacuoles present which were shown to contain glycogen by the PAS and PAS diastase reaction. Quantitative glycogen determination and enzyme assays were not done. He was discharged with a diagnosis of “Glycogen Storage Disease.”
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Over the ensuing years he was treated with frequent feedings. He had weekly episodes of vomiting occurring in the morning if he drank milk, and at other times when he became excited or angry. He also had frequent episodes of otitis and tonsillitis. The mother reported no convulsions or muscle weakness in the child. In August 1968 at age 6% years the patient was re-admitted to Children’s Hospital for further investigation. At this time it was noted that his developmental milestones were normal and that he was a “superior” student in school. His physical examination revealed normal vital signs. His height was 112 cm. (tenth percentile), weight 21.2 Kg. (25th percentile), head circumference 54 cm. (97th percentile), chest circumference 61 cm. (90th percentile), and abdominal girth 67.5 cm. (> 97th percentile). A grade II/VI apical systolic murmur was heard and it radiated widely. His liver was enlarged to 10 cm. below the right costal margin; it was nontender, smooth and had a rounded edge. The remainder of his physical examination was unremarkable. Admission laboratory studies, including hemoglobin and hematocrit, white blood count and differential, bleeding time, clotting time, BUN, electrolytes, SGOT, SGPT, serum protein electrophoresis and blood gases were all normal. A single urinalysis at the time of admission revealed 3 plus acetonuria, but was otherwise normal. Skeletal survey revealed a bone age of four years, which was retarded for his chronological age. Blood sugars obtained four hours after meals were 92 mg. per cent, 80 mg. per cent, 79 mg. per cent and 87 mg. per cent. An oral GTT (1.8 Gm. glucose/Kg.) revealed a fasting blood sugar of 66 mg. per cent, at 30 min. 148 mg. per cent, 60 min. 162 mg. per cent, 120 min. 124 mg. per cent, 180 min. 59 mg. per cent, 240 min. 58 mg. per cent and at 300 min. 67 mg. per cent. A glucagon tolerance test (0.03 mg. glucagon/Kg.. subcutaneously) gave the following results: fasting blood glucose 67 mg. per cent, 10 min. 80 mg. per cent, 20 min. 96 mg. per cent, 30 min. 113 mg. per cent, 45 min. 117 mg. per cent, 60 min. 104 mg. per cent. 90 min. 79 mg. per cent and 120 min. 70 mg. per cent. Liver and muscle biopsies were performed on the patient, and the results of biochemical analysis are tabulated in Table I.
METHODS AND MATERIALS The methods for assay of leukocyte phosphorylase,” hepatic and muscle phosphorylase,r hepatic glucose-6-phosphatase ,r hepatic and muscle debranching enzyme,7 and hepatic and muscle alpha-glucosidases have been previously described. The methods for assay of glycogen in liver,? muscle,9 and erythrocytesla are also described elsewhere. Previously Williams and Field reported normal leukocyte phosphorylase to be 29.6 + 1.2 (SEM) pg. PO,/107 wbc/30 min. However, using the exact procedure we have found leukocyte phosphorylase activity to be 73.6 t 3.0 (SEM) &g. PO,/107 wbc/30 min. in a series of 26 normals. The range was from 52.0 to 106.8. The reason for the difference between these two normal series is not apparent. Esmann et al, 11 however, utilizing this same assay, recently reported activities of leukocyte phosphorylase in normals with values similar to ours (73.28 ? 1.09 (SEM) pg. PO,/107 wbc/30 min.). The reliability of the erythrocyte glycogen assay was tested by determining the recovery of 100 fig. of glycogen added to aliquots of packed erythrocytes. The recoveries ranged from 19 per cent to 35 per cent. The results were not corrected for recovery. RESULTS
The diagnosis of Type VI GSD in the patient was established by the clinical findings and by the marked reduction in liver phosphorylase (20 min. 12.8 mg. POJGm. protein and 30 min. 18.0 mg. POJGm. protein) and in leukocyte phosphorylase 35.98 PGm. P04/107wbc/30 min.). Liver glycogen was increased (7.5% ), whereas muscle glycogen content was normal (0.5% ). Other liver and muscle enzymes concerned with glycogen metabolism were normal. Leukocyte phosphorylase and erythrocyte glycogen values in the patient and eleven of his relatives are summarized in Table 2 and the pedigree illustrated in Fig. 1. The patient was the only member of his family with signs and symptoms
Debranching (14C method) Glycogen alpha-Glucosidase Phosphorylase
Debranching (14C method) Phosphorylase Glycogen
Liver Muscle Muscle Muscle
Muscle WBC RBC
wbc: white blood cells. rbc: red blood cells. cpm: counts per minute.
Glycogen alpha-Glucosidase Glucose-6-phosphatase Phosphorylase
Assay
Liver Liver Liver Liver
Tissue
7.5% 6.89 24 12.8 (20 min.) and 18.0 (30 min.) 1,340,000 0.5% 0.90 263 (20 min.) and 362 (30 min.) 1,000,000 35.98 60.25
Results
Table 1 .-Enzyme
Assays (Mean C SEM)
up to 5% 12.93 t 2.37 w moles glucose liberated/min./mg. protein 26.7 * 5.1 mg. phosphorus liberated/Gm. protein 72.0 f 14.7 (20 min.) and 81.0 t 18.9 (30 min.) mg. phosphorus liberated/ Gm. protein 1,123,OOO k 435,000 cpm/Gm. protein up to 1.0% 1.74 k 41 mp moles glucose liberated/min./mg. protein 144 f 21 (20 min.) and 220 + 37 (30 min.) mg. phosphorus liberated/Gm. protein 1,307,OOO + 103,000 cpm/Gm. protein 73.61 -t 3.01 pg. PO,/107 wbc/30 min. 23.59 k 5.94 ,eg. glycogen/Gm. Hgb.
Normals
and Special Studies on Patient T.R.
N P
? $
u i;3
F $
SCHWARTZ
242
ET AL.
Table 2.-Leukocyte Phosphorylase Activities and Eerythrocyte Glycogen Levels in Normal Subjects, Patient T.R., and Members of His Family ___~~_. Erythrocyte Glycogen .__@g. GlycogedGm. Hgb.
Leukocyte Phosphorylase Person
Patient (T.R.) Brother (J.R.) Mother (CR.) Father (C.L.R.) Grandmother (M.R.C.) (maternal) Grandfather (R.C.) (maternal) Grandmother (H.R.) (paternal) Uncle, (L.C.) (maternal) Uncles (J.C.) (maternal) Aunt (G.S.) (paternal) Grandaunt (E.G.) (maternal) Granduncle (J.A.C.) (maternal) Mean Standard Error of the Mean Normals Range Mean Standard Error of the Mean *Number
____~~~ k%. PO+/lO’wbc/30 min.
35.98 33.68 25.26 45.12 50.68 47.20 44.25 22.31 24.51 44.36 72.20 74.96 43.38 4.91 (n = 26) 52.01-106.81 73.61 3.02
60.25
(2)* (1) (2) (1) (4) (2) (1) (2) (2) (1) (1) (2)
(l)*
117.56 (2) 39.49 (1) 33.30 (3) 13.66 (2) 42.42 (1) 20.33 (2) 66.51 (2) 71.65 (1) 22.49 (1) 48.77 9.89 (n = 21) 3.91-133.16 23.59 5.94
of determinations.
of glycogen storage disease. The patient’s leukocyte phosphorylase activity determined on two occasions averaged 35.98 pg. POa/107wbc/30 min., well outside our normal range. Both the mother’s leukocyte phosphorylase activity (25.26 pg. P01/107wbc/30 min.) and the father’s leukocyte phosphorylase activity (45.12 pg. P0,/107wbc/30 min.) were reduced. The patient’s brother had a leukocyte phosphorylase activity similar to that of the patient (33.68 pg. POJlO’wbc/30 min.). He was asymptomatic and did not have hepatomegaly. The patient’s maternal grandmother had a leukocyte phosphorylase activity just below our normal range 50.68 pg. P01/107wbc/30 min.). The maternal grandfather’s leukocyte phosphorylase activity (47.2 pg. PO,/lOrwbc/ 30 min.) was also outside our normal range. The only paternal grandparent studied was the paternal grandmother. Her leukocyte phosphorylase activity was 44.25 pg. P0+/107wbc/30 min., which also falls below our normal range. Two maternal uncles and one paternal aunt had low leukocyte phosphorylase activities. The maternal grandfather’s brother and sister had normal activities of this enzyme. The patient’s leukocyte phosphorylase activity did not differ significantly from the activities found in other family members whose values fell below our normal range. Table 2 also includes the results of the erythrocyte glycogen studies. The 2 1 normal subjects had values ranging from 3.91 pg. glycogen/Gm. Hgb to 133.16 pg. glycogen/Gm. Hgb, with a mean of 23.59 pg. glycogen/Gm.Hgb. The patient’s erythrocyte glycogen (60.3 pg./Gm. Hgb) and the erythrocyte glycogen levels of all members of his family tested were within our normal range. DISCUSSION
This patient provides another
example of reduced
hepatic
and leukocyte
GLYCOGEN
STORAGE
243
DISEASE
EG.
7220
-
7%
Cs.
44.36
Demo& LeukocyteFt-qhcfyhse DecreasedLeuk and Clinkd hkZ%Eb~yk Type VI GSD
q=Male o=Femde NT= Not Tested Fig. 1 .-Pedigree
of family studied.
phosphorylase activity found in patients with Type VI GSD. Similar observations of reduced leukocyte phosphorylase activity in patients with this disease have been made by other workers.2-4*6J1J2 In the present family reduced leukocyte phosphorylase activity was found in the patient’s mother and father, brother, the paternal grandmother, maternal grandparents, two maternal uncles and a paternal aunt, none of whom have clinical evidence of glycogen storage disease. A sex-linked mode of inheritance for this disease is unlikely because the patient’s brother, father, maternal grandfather, and maternal uncle have low leukocyte phosphorylase activities and no clinical evidence of glycogen storage disease. A dominant mode of inheritance for this disease is also unlikely since none of the relatives of the patient who have low leukocyte phosphorylase activities have any clinical evidence of Type VI GSD. Therefore, an autosomal recessive mode of inheritance is most likely. This hypothesis is supported by the finding of low leukocyte phosphorylase activities in clinically normal relatives of both sexes on either side of the family, this finding being the only abnormality manifested by the heterozygous individual. Both an autosomal recessive and a sex-linked inheritance have been postulated for Type VI alycogen storage disease. Williams and Field* in 1961 re-
244
SCHWARTZ
ET AL.
ported decreased activities of leukocyte phosphorylase in two brothers with Type VI GSD. Leukocyte phosphorylase activity in the patient’s mother was intermediate between normal values and those of her sons while the father was normal. Similar findings were reported by Htilsman et al.” in a family they studied. The findings of these two groups of investigators suggest a dominant mode of inheritance, either sex-linked or autosomal. Hers,l however, in his original description of this disease, reported two females with clinical and laboratory findings of Type VI GSD whose parents had no clinical evidence of glycogen storage disease, thus mitigating against a sex-linked mode of inheritance. Wallis et a1.4 found markedly decreased leukocyte phosphorylase activity in a patient with Type VI GSD and levels intermediate between those of the patient and normal subjects in both parents, the maternal grandmother and the maternal grandmother’s mother, all of whom were clinically normal. On the basis on these results it was concluded that this family demonstrated an autosomal recessive mode of inheritance. Esmann et al.ll found low leukocyte phosphorylase levels in a patient with Type VI GSD, his brother, mother. maternal grandfather and a distant male cousin on the maternal side, whereas the father had normal leukocyte phosphorylase activity. The authors suggested a sex-linked mode of inheritance. HuijingO studied leukocyte phosphorylase and leukocyte phosphorylase kinase activities in 11 patients with Type VI GSD. Ten of these patients (9 males and 1 female) had low activities of leukocyte phosphorylase kinase (0 to 60% of the lowest normal values). Five mothers of the children with phosphorylase kinase deficiency were tested; four had low leukocyte phosphorylase kinase activities. Three fathers were also tested and found to have normal leukocyte phosphorylase kinase activities. Two of these three fathers were from families in which the mother’s leukocyte phosphorylase kinase was found to be low. He suggested that these results were best explained by postulating a sex-linked mode of inheritance in the form of Type VI GSD associated with a deficiency of leukocyte phosphorylase kinase (Type VI A) .13 The eleventh patient had low leukocyte phosphorylase activity, but normal leukocyte phosphorylase kinase activity. The mother and a sib of the patient also had normal leukocyte phosphorylase kinase activity. Both the mother and father of this patient, however, had low leukocyte phosphorylase activities suggesting an autosomal recessive mode of inheritance in the form of Type VI GSD associated with a deficiency of leukocyte phosphorylase in the presence of an intact activating system (Type VI B) .I3 Since our patient’s family appears to demonstrate an autosomal recessive pattern, he probably has a deficit of phosphorylase with an intact activating system. Huijing” noted that the addition of 1 mM AMP restored to normal leukocyte phosphorylase activity of patients with leukocyte phosphorylase kinase deficiency. Although leukocytes from our patient were assayed in a medium containing 0.8 mM AMP (close to that used by Huijing). phosphorylase activity was still significantly decreased. This suggests that our patient has a deficiency of phosphorylase itself with an intact activating system. It was not possible to assay leukocytes of our patient and his relatives for phosphorylase kinase. In addition, assay of leukocyte phosphorylase with and without added AMP would be of value.
245
GLYCOGEN STORAGE DISEASE
Although Wallis et al.* felt that elevations of erythrocyte glycogen values were a sensitive index of the carrier state of Type VI GSD, our results were not consistent with this conclusion. They found elevated erythrocyte glycogen levels in their patient, both parents, the maternal grandmother, the maternal grandmother’s mother and in six other members of both sides of the family. However, in our patient and his relatives erythrocyte glycogens were normal. The poor recoveries of glycogen obtained using this determination could certainly be a major factor in the apparent uselessness of this procedure in our hands. SUMMARY
A child with Type VI GSD, confirmed by biopsy, and 12 members of his immediate family have been studied utilizing leukocyte phosphorylase activities and erythrocyte glycogen levels to further elucidate the pattern of inheritance of this disease. Leukocyte phosphorylase activities were low in the patient and his clinically normal brother, parents, maternal grandparents, paternal grandmother, two maternal uncles and a paternal aunt. The data are consistent with an autosomal recessive mode of inheritance in this family. Erythrocyte glycogen studies were not useful in defining the mode of inheritance of this disease. The data are discussed in terms of the two modes of inheritance and the two enzymatic defects which have been described in this disease. ACKNOWLEDGMENTS We would like to thank Dr. Mark Steele for his helpful comments of this paper.
during the preparation
REFERENCES 1. Hers, H. G.: Etudes enzymatiques sur fragments hepatiques. Rev. Intern. Hepatol. 12:35, 1959. 2. Williams, H. E., and Field, J. B.: Low leukocyte phosphorylase in hepatic phosphorylase deficient glycogen storage disease. J. Clin. Invest. 40:1841, 1961. 3. Hiilsmann, W. C., Oei, T. L., and van Creveld, S.: Phosphorylase activity in leukocytes from patients with glycogen storage disease. Lancet 2:581, 1961. 4. Wallis, P. G., Sidbury, J. B., and Harris, R. C.: Hepatic phosphorylase defect: studies on peripheral blood. Amer. I. Dis. Child. 111:278, 1966. 5. Hug, G., Garancis, I. C., Schubert, W. K., and Kaplan, S.: Glycogen storage disease, Types II, III, VII, and IX. Amer. J. Dis. Child., 111:457, 1966. 6. Huijing, F.: Enzymes of glycogen metabolism in leukocytes in relation to glycogen-storage disease. Control of GlyUniversitetsforlaget, cogen Metabolism, 1968, p. 115. 7. Field, J. B., Epstein, S. M., and Egan, T.: Studies in glycogen storage disease. I.
Intestinal glucose-6-phosphatase activity in patients with Von Gierke’s disease and their parents. I. Clin. Invest. 44:1240 ,1965. 8. Williams, H. E.: Alpha-glucosidase in human leukocytes. Biochem. Biophys. Acta. 124~34, 1966. 9. Caroll, N. V., Longley, R. W., and Roe, J. H.: The determination of glycogen in liver and muscle by use of the anthrone reagent. J. Biol. Chem. 220:583, 1956. 10. Williams, H. E., Kendig, E. M., and Field, J. B.: Leukocyte debranching enzyme in glycogen storage disease. J. Clin. Invest. 42:656, 1963. 11. Esmann, V., Hobolth, N., and Jorgensen, J. I.: Heredity of leukocyte phosphorylase and amylo-l , 6-glucosidase deficiency. J. Pediat. 74:90, 1969. 12. van Creveld, S., and Huijing, F.: Glycogen storage disease. Biochemical and clinical data in sixteen cases. Amer. J. Med. 38:554, 1965. 13. van Creveld, S., and Huijing, F.: Glycogenosis. XII Congreso International de Pediatria II, 1968, pp. 554-572.